Masao Yoshinari

Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts

This study investigated the influence of surface wettability on competitive protein adsorption and the initial attachment of osteoblasts. A thin-film coating of hexamethyldisiloxane (HMDSO) and subsequent O(2)-plasma treatment was carried out on substrates with a mirror surface in order to create a wide range of wettabilities. The adsorption behavior of fibronectin (Fn) and albumin (Alb) in both individual and competitive mode, and the initial attachment of mouse osteoblastic cells (MC3T3-E1) over a wide range of wettabilities were investigated. The contact angle of HMDSO coatings without O(2)-plasma treatment against double-distilled water was more than 100 degrees, whereas it dramatically decreased after the O(2)-plasma treatment to almost 0 degrees, resulting in super-hydrophilicity. Individually, Fn adsorption showed a biphasic inclination, whereas Alb showed greater adsorption to hydrophobic surfaces. In the competitive mode, in a solution containing both Fn and Alb, Fn showed greater adsorption on hydrophilic surfaces, whereas Alb predominantly adsorbed on hydrophobic surfaces. The initial attachment of osteoblastic cells increased with an increase in surface wettability, in particular, on a super-hydrophilic surface, which correlated well with Fn adsorption in the competitive mode. These results suggest that Fn adsorption may be responsible for increasing cell adhesion on hydrophilic surfaces in a body fluid or culture media under physiological conditions.

The attachment and growth behavior of osteoblast-like cells on microtextured surfaces.

In previous studies, we showed that the application of microgrooves on a surface can direct cellular morphology and the deposition of mineralized matrix of osteoblast-like cells (Biomaterials 20 (1999) 1293; Clin. Oral Impl Res. 11 (2000) 325). In this study, we evaluated the attachment and growth behavior of these cells, using scanning- and transmission electron microscopy (SEM/TEM). Smooth and microgrooved polystyrene substrates were made (groove depth 0.5-1.5 microm, groove- and ridge width 1-10 microm). On these substrates, osteoblast-like cells were cultured for periods up to 16 days. SEM showed that the cells, and their extensions, closely followed the surface on smooth and wider grooved (>5 microm) substrates. In contrast, narrow grooves (<2 microm) were bridged. After 16 days of incubation, the matrix showed extensive deposition of collagen fibrils, and the formation of calcified nodules. With TEM it was shown that on the smooth and wider grooved substrates, focal adhesions were spread throughout the surface. However, on narrow grooves focal adhesions were always positioned on the edges of surface ridges only. Apparently, most extracellular matrix (ECM) was produced by the cells that directly adhered to the substrate. Deposition of ECM was seen in the surface grooves, as well as in between the cell layers. On basis of the current study and previous experiments, we conclude that microgrooves are able to influence bone cell behavior by (1) determining the alignment of cells and cellular extensions, (2) altering the formation and placement of cell focal adhesions, and (3) altering ECM production. Therefore, microgrooved surfaces seem interesting to be applied on bone-anchored implants.

Substrate affects the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2).

Interaction between implant materials and bone cells contributes to the clinical success of dental implants. The object of this study was to investigate the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2) to pure titanium (Ti), hydroxyapatite (HA), and glass. We, therefore, performed a time-course study for examining the area, attachment rate, distribution of focal adhesion kinase (FAK) vinculin, and actin, and the motility of Saos-2 cells on the materials. On Ti, cell area increased gradually, whereas on HA, cells spread quickly, but quitted spreading at 12 h after cell seeding. The number of cells on HA was greater than on the other materials. On Ti, the numbers of FAK- and vinculin-positive focal adhesions increased continuously. On HA, although the number of FAK-positive focal adhesions also increased continuously, the number of vinculin-positive focal adhesions decreased. Furthermore, actin staining showed that the cells on HA poorly formed stress fibers with weak polarity, whereas the cell on Ti possessed well-defined polarized stress fibers. On HA, cells started extension earlier than on Ti, motility was inactive, and the cells settled on the materials. These results suggest that the earlier settling of osteoblasts on HA might result in earlier osteogenesis on HA than other materials.

Characterization and protein-adsorption behavior of deposited organic thin film onto titanium by plasma polymerization with hexamethyldisiloxane.

Plasma polymerized hexamethyldisiloxane (HMDSO) thin film was deposited onto titanium using a radio-frequency apparatus for the surface modification of titanium. A titanium disk was first polished using colloidal silica at pH=9.8. Plasma-polymerized HMDSO films were firmly attached to the titanium by heating the titanium to a temperature of approximately 250 degrees C. The thickness of the deposited film was 0.07-0.35mum after 10-60min of plasma polymerization. The contact angle with respect to double distilled water significantly increased after HMDSO coating. X-ray photoelectron spectroscopy revealed that the deposited thin film consisted of Si, C, and O atoms. No Ti peaks were observed on the deposited surface. The deposited HMDSO film was stable during 2-weeks immersion in phosphate buffer saline solution. Fourier transform reflection-absorption spectroscopy showed the formation of Si-H, Si-C, C-H, and Cz.dbnd6;O bonds in addition to Si-O-Si bonds. Quartz crystal microbalance-dissipation measurement demonstrated that the deposition of HMDSO thin films on titanium has a benefit for fibronectin adsorption at the early stage. In conclusion, plasma polymerization is a promising technique for the surface modification of titanium. HMDSO-coated titanium has potential application as a dental implant material.

Prevention of biofilm formation on titanium surfaces modified with conjugated molecules comprised of antimicrobial and titanium-binding peptides

Specific binding of antimicrobial peptides to titanium (Ti) surfaces may serve to prevent biofilm formation, leading to a reduction in peri-implantitis. This study evaluated the binding behavior of conjugated molecules consisting of antimicrobial and hexapeptidic Ti-binding peptides (minTBP-1) using the quartz crystal microbalance (QCM-D) technique, and investigated the effect of modification of Ti surfaces with these peptides on the bioactivity of Porphyromonas gingivalis. Four kinds of peptide were prepared: histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), minTBP-1 + histatin 5 (RKLPDAPDSHAKRHHGYKRKFHEKHHSHRGY), lactoferricin (FQWQRNMRKVR), and minTBP-1 + lactoferricin (RKLPDAPGGFQWQRNMRKVR). The QCM-D analysis demonstrated that significantly larger increases in peptide adsorption were observed in the conjugated peptides than in antimicrobial peptides alone. In addition, ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control. These results indicate that surface modification with conjugated molecules consisting of antimicrobial and Ti-binding peptides is a promising method for reduction of biofilm formation on Ti surfaces.

An immunoelectron microscopic localization of noncollagenous bone proteins (osteocalcin and osteopontin) at the bone–titanium interface of rat tibiae

This study was designed to investigate by postembedding immunogold method the localization and distribution of osteocalcin (Ocl) and osteopontin (Opn) at the bone-titanium interface in rat tibiae 14 and 28 days postimplantation to determine which bone proteins are present at this interface. Both proteins were widely distributed on the newly formed bone and accumulated predominantly in the region of bone close to the titanium, in electron-dense patches in the bone, and at the osteocytic lacunae. Collagenous osteoid showed little or no labeling for either Ocl or Opn. An amorphous zone (20-50 nm) was interposed between the titanium and interfacial slender cells, osteoid, or bone, and was labeled strongly for Ocl but only weakly for Opn. Furthermore, a second electron-dense layer, the lamina limitans, which faces the titanium, was labeled strongly for Opn but weakly for Ocl. Ocl as a marker protein of osteoblasts was sometimes found in the granules and vesicles of the interfacial cells and extracellularly in their intercellular spaces, close to the titanium. However, Opn was not detected in any granules. This is the first report to show that the amorphous zone contains large amounts of Ocl and small amounts of Opn, and that bone contacts titanium through this Ocl-rich amorphous zone. Furthermore, it is suggested that the interfacial cells seem to be osteoblasts, and that Ocl in the amorphous zone is produced and secreted by these cells and functions with Opn as a regulator of the mineralization front close to the titanium, and as a mediator of cell-matrix and matrix-matrix/mineral adhesion along the titanium.

Influence of surface topography and surface physicochemistry on wettability of zirconia (tetragonal zirconia polycrystal)

Surface modification technologies are available for tetragonal zirconia polycrystal (TZP) to enhance its bioactivity and osseointegration capability. The surface wettability of an implant material is one of the important factors in the process of osseointegration, possibly regulating protein adsorption, and subsequent cell behavior. The aim of this study was to clarify the effect of topographical or physicochemical modification of TZP ceramics on wettability to determine the potential of such treatment in application to implants. Several types of surface topography were produced by alumina blasting and acid etching with hydrofluoric acid; surface physicochemistry was modified with oxygen (O(2)) plasma, ultraviolet (UV) light, or hydrogen peroxide treatment. The obtained specimens were also subjected to storage under various conditions to evaluate their potential to maintain superhydrophilicity. The results showed that surface modification of surface topography or physicochemistry, especially of blast/acid etching as well as O(2) plasma and UV treatment, greatly increased the surface wettability, resulting in superhydrophilicity. X-ray photoelectron spectroscopy revealed that a remarkable decrease in carbon content and the introduction of hydroxyl groups were responsible for the observed superhydrophilicity. Furthermore, superhydrophilicity was maintained, even after immersion in an aqueous solution, an important consideration in the clinical application of this technology.

In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue.

Many studies have examined mineralized-tissue formation on titanium in vivo and in vitro; however, no precise method for measuring the mineralized tissue produced by cultured osteoblastic cells on titanium in vitro has been established. This study developed a method for measuring mineralized-tissue formation by cultured rat osteoblastic cells on titanium in vitro, and re-evaluated the effects of modifying the titanium surface. We used calcein blue, which binds to mineralized tissue, and measured the resulting fluorescence under ultraviolet light. A 1-h incubation with 3.1x10(-3)M calcein blue resulted in sufficient fluorescence of bone-like nodules on the titanium. Consequently, we found that osteoblastic cells produced larger bone-like nodules on titanium than in culture dishes. Fewer bone-like nodules formed on calcium-ion impregnated titanium than on pure titanium. However, when bisphosphonate was immobilized on the calcium-ion impregnated titanium, more bone-like nodules formed than on pure titanium. The results suggest that bisphosphonate immobilization on titanium is useful for stimulating mineralized-tissue formation on titanium implants.

Motif-programmed artificial extracellular matrix.

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.

Corrosion mechanism of Ti–Cr alloys in solution containing fluoride

OBJECTIVE The objective of this study was to clarify the influence of chromium content on surface reaction of Ti-Cr alloys in an acidic fluoride-containing saline solution. METHODS Four Ti-Cr alloys containing 5, 10, 15 or 20 mass% chromium were characterized in terms of dissolution of metals in an acidic fluoride-containing saline solution and surface structure by X-ray photoelectron spectroscopy and Auger electron spectroscopy. RESULTS Total amount of metals dissolved from each alloy decreased with increase in chromium content. The surface oxide films of Ti-Cr alloys before and after immersion in an acidic fluoride-containing saline solution consisted of titanic and chromic species, such as oxide, hydroxide, and hydrate. The [Cr]/([Ti]+[Cr]) ratio in the surface oxide film on as-polished Ti-Cr alloys was closely correlated with chromium content. However, the ratio in any alloy approximately doubled after immersion. Although thick oxide films were observed after immersion, all alloys showed a thinner oxide film than commercially pure titanium. SIGNIFICANT In all alloys, concentration of chromic species such as oxide and hydroxide in the surface oxide film was associated with chromium content, and chromic species improved corrosion resistance to fluoride.